K562 genomic DNA was bisulfite converted using the EpiTect Fast DNA Bisulfite Kit, and a 100 bp amplicon from the X-chromosome was amplified using cytosine-free primers to allow amplification of both unconverted and bisulfite converted DNA. PCR products were cloned into a vector and Sanger sequencing was done to identify converted cytosines. 1252 individual cytosines were analyzed, revealing a highly efficient cytosine conversion rate of 99.8%. This high conversion rate is especially significant as the genomic DNA was derived from a female; therefore, one of the 2 X-chromosomes is highly methylated.|Streamlined and straightforward, the EpiTect Fast 96 DNA Bisulfite Kit enables complete bisulfite conversion and cleanup of genomic DNA in approximately 1 hour.|Upon completing deparaffinization, lysis, and decrosslinking steps, the bisulfite conversion is completed using the included EpiTect Fast 96 DNA Bisulfite Kit.|Upon completion of the sample lysis steps, bisulfite conversion is completed using the included EpiTect Fast 96 DNA Bisulfite Kit.|Following bisulfite conversion with the EpiTect Fast DNA Bisulfite Kit, 1 µg of genomic DNA showed the lowest level of fragmentation, with a size distribution ranging from several hundred base pairs to over 6 kb, compared to other methods with peaks at 500 bp and 1 kb, respectively. Samples were analyzed on an Agilent RNA 6000 Nano Chip.|Following bisulfite conversion of genomic DNA using the EpiTect Fast DNA Bisulfite Kit or a kit from Supplier Z, unconverted genomic DNA and bisulfite converted DNA were coamplified and then sequenced by Pyrosequencing to quantify the rate of conversion of individual cytosines. [A] The EpiTect Fast DNA Bisulfite Kit gave comparable results to a kit from Supplier Z when 2 µg of DNA was analyzed. [B] However, when a limited amount of DNA was analyzed (10 ng), the 4 cytosine residues shown in this Pyrogram demonstrate that the EpiTect Fast DNA Bisulfite Kit delivered significantly better results than the kit from Supplier Z. (Percentage of each cytosine residue that did not undergo bisulfite conversion using the kit from Supplier Z vs. the EpiTect Fast DNA Bisulfite Kit: 12% vs. 2%, 5% vs. 1%, 37% vs. 9% and 11% vs. 2%, respectively.)|Upon addition to the bisulfite conversion reaction, the DNA Protect Buffer should turn from green to blue, indicating sufficient mixing and correct pH.||Depending on sample type, the bisulfite conversion of DNA calls for the use of a specific kit and protocol. Kits contain buffers optimized for DNA extraction and all reagents for the DNA bisulfite conversion.|

Highly efficient bisulfite conversion

Bisulfite treatment of DNA and the resulting conversion of unmethylated cytosines to uracils is the most critical step in methylation analysis, since the reaction efficiency will greatly impact the reliability of downstream analyses. EpiTect Fast Bisulfite Conversion Kits contain innovative Bisulfite Solution, which enables ultrafast, highly efficient bisulfite conversion, even on difficult-to-analyze samples such as X-chromosome genomic DNA derived from a female sample, where one of the 2 X-chromosomes is highly methylated (see figure "Highly efficient cytosine conversion of X chromosome DNA"). Quantification of individual cytosine conversion by Pyrosequencing shows that significantly better conversion is achieved with the EpiTect Fast Bisulfite Conversion Kit than a kit from another supplier, particularly when the amount of starting material is limited (see figure "Superior bisulfite conversion").

Reduced DNA fragmentation

The harsh conditions necessary for complete bisulfite conversion usually lead to a high degree of DNA fragmentation and sample loss, leading to low DNA yield, highly fragmented DNA, and irreproducible conversion rates. QIAGEN's unique DNA Protect Buffer prevents DNA degradation, resulting in fully-converted DNA of much larger fragment sizes than DNA converted with other suppliers' kits (see figure "Analysis of DNA fragment sizes after bisulfite conversion").

Principle

Fast and complete bisulfite conversion of sample DNA is essential for accurate DNA methylation analysis. EpiTect Fast 96 Bisulfite Conversion Kits include an innovative Bisulfite Solution that allows highly efficient bisulfite conversion in as little as 30 minutes. Streamlined sample lysis and conversion protocols enable efficient purification and bisulfite conversion of DNA, while unique DNA Protect technology minimizes DNA fragmentation during bisulfite treatment. The 3 EpiTect Fast 96 Bisulfite Conversion Kits are interconnected, functioning as an integrated system of solutions for direct bisulfite conversion of DNA from a broad range of starting materials (see figure "Interconnected protocols of the EpiTect Fast 96 Bisulfite Conversion Kits"). However, each kit has unique features, addresses a specific sample type, and includes all buffers and reagents necessary to function independently. The components in each kit combine to deliver effective lysis of samples, minimal loss of DNA during lysis, complete bisulfite conversion, robust protection during conversion, and thorough cleanup of DNA.

Fast bisulfite conversion

EpiTect Fast 96 Bisulfite Conversion Kits provide a very fast and streamlined procedure for efficient conversion and purification of DNA prepared from genomic DNA, FFPE, blood, cells, or tissue samples. The kits contain highly concentrated Bisulfite Solution, which reduces the time required to convert unmethylated cytosine residues into uracil from several hours to as little as 30 minutes, as well as preparation buffers that make it unnecessary to isolate the DNA prior to bisulfite treatment. Furthermore, the bisulfite thermal cycling program provides an optimized series of incubation steps necessary for thermal DNA denaturation and subsequent sulfonation and cytosine deamination, enabling high cytosine conversion rates of over 99%. Desulfonation, the final step in chemical conversion of cytosines, is achieved by a convenient step that is included in the purification procedure.

DNA Protect Buffer

Fast and complete bisulfite conversion of sample DNA is the most critical step for the correct determination of a methylation pattern. This is achieved by incubating the DNA in high bisulfite salt concentrations at high temperature and low pH. These harsh conditions often lead to a high degree of DNA fragmentation and subsequent loss of DNA during purification. Common bisulfite procedures usually require high amounts of input DNA to compensate for DNA degradation during conversion and DNA loss during purification, which often lead to low DNA yield, highly fragmented DNA, and irreproducible conversion rates. With EpiTect Fast 96 Bisulfite Conversion Kits, DNA fragmentation is prevented during bisulfite conversion by the unique DNA Protect Buffer, which is formulated to prevent the fragmentation usually associated with bisulfite treatment of DNA at high temperatures and low pH values. It also provides effective DNA denaturation, resulting in the single-stranded DNA necessary for complete cytosine conversion. DNA Protect Buffer contains a convenient pH indicator dye as a mixing control allowing confirmation of the correct pH for cytosine conversion (see figure "DNA Protect Buffer").

Deparaffinization of FFPE samples

The first step in extracting DNA from FFPE samples is deparaffinization, whereby paraffin is first dissolved and then removed, exposing samples for subsequent treatment with lysis buffer and proteinase K. With the EpiTect Fast 96 FFPE Bisulfite Kit, the deparaffinization, lysis, and decrosslinking of FFPE slices is optimized with Deparaffinization Solution, an innovative chemistry that facilitates deparaffinization with minimal handling. Deparaffinization Solution contributes to high DNA recovery rates, since it remains on the sample while proteinase K digestion is carried out. With no need to pellet the FFPE sample or to remove the paraffin-containing supernatant, the risk of sample loss during deparaffinization is avoided.

Procedure

EpiTect Fast 96 Bisulfite Conversion Kit procedures comprise a few simple steps: preparation of DNA from a sample, bisulfite-mediated conversion of unmethylated cytosines; binding of the converted single-stranded DNA to the membrane of an EpiTect 96 Plate; washing; desulfonation of membrane-bound DNA; washing of the membrane-bound DNA to remove the desulfonation agent; and elution of the pure, converted DNA from the 96-well plate. Sample preparation is different for FFPE slices, whole blood, cell cultures, or tissues, whereas the procedure for bisulfite conversion of extracted DNA is the same for all sample types (see figure "Interconnected protocols of the EpiTect Fast 96 Bisulfite Conversion Kits"). All protocols achieve the same cytosine conversion rates and lead to equal DNA recoveries after purification of converted DNA, independent of DNA starting amounts.

Kit descriptions

EpiTect Fast 96 DNA Bisulfite Kit

The EpiTect Fast 96 DNA Bisulfite Kit includes carefully formulated buffers and reagents to provide streamlined and effective bisulfite conversion of genomic DNA and subsequent cleanup of the converted DNA (see Figure "EpiTect Fast 96 DNA Bisulfite Kit procedure"). The novel Bisulfite Solution enables complete bisulfite conversion in as little as 30 minutes, while DNA Protect technology and EpiTect 96 Plates allow the generation of fully converted DNA with minimal degradation using either a centrifuge or vacuum manifold. The EpiTect Fast 96 DNA Bisulfite Kit can be used for conversion of 1 ng–2 µg of DNA.

EpiTect Fast 96 FFPE Bisulfite Kit

Formalin-fixed, paraffin-embedded (FFPE) tissues are processed with the EpiTect Fast 96 FFPE Bisulfite Kit, which consists of the EpiTect Fast 96 FFPE Lysis Kit, containing specialized buffers for efficient deparaffinization and lysis of FFPE tissue slices (see figure "EpiTect Fast 96 FFPE Bisulfite Kit procedure"), and the EpiTect Fast 96 DNA Bisulfite Kit for fast and effective bisulfite conversion of the extracted DNA. The protocol includes an optimized step to facilitate binding of DNA and can be used with single slices of FFPE tissue (10 µm in thickness). The DNA fragmentation that inevitably occurs when DNA is extracted from FFPE tissues is made worse by the harsh conditions of subsequent bisulfite treatment. QIAGEN's reliable DNA Protect technology minimizes fragmentation during the conversion reaction, resulting in superior yields of bisulfite converted DNA.

EpiTect Fast 96 LyseAll Bisulfite Kit

Whole blood, cultured cells, or tissue samples are processed with the EpiTect Fast 96 LyseAll Bisulfite Kit, which consists of the EpiTect Fast 96 LyseAll Lysis Kit (see figure "EpiTect Fast 96 LyseAll Bisulfite Kit procedure") with an innovative lysis buffer and the EpiTect Fast 96 DNA Bisulfite Kit for bisulfite conversion of the extracted DNA. The protocol includes an optimized step to facilitate binding of DNA along with uniquely formulated lysis buffers to streamline the release of DNA from samples. This kit can be used with 0.5–20 µl blood or 10–105 cells (as little as 60 pg of DNA).

Applications

EpiTect Fast 96 Bisulfite Conversion Kits deliver fast and efficient bisulfite conversion of DNA from various starting material that is highly suited for all techniques currently used for the analysis of DNA methylation, including: